Long QT syndrome (LQTS) increases the risk of torsades de pointes, a ventricular arrhythmia that can degenerate into ventricular fibrillation and sudden death. LQTS patients are currently treated with -adrenergic blockers; however, failure of -blocker therapy is significant in young children and women. ICD therapy has been recommended for this subset of high-risk LQTS patients; however, devices are expensive and not available to all patients in need. Thus, there remains a need for the discovery and development of additional treatment options for LQTS. Mechanism-based pharmacotherapy (e.g., mexilitine for LQT3) has been explored in the past, but little is known about the mechanisms of action or therapeutic utility of the recently discovered hERG1 channel activators. hERG1 activators shorten cardiac action potentials by altering channel gating, either by suppression of P-type inactivation, slowing deactivation, increasing channel open probability or a combination of these effects. We have mapped the putative binding site for several known hERG1 activators and correlated their binding site to primary mechanism of action. Here we propose to define the structural basis of altered channel gating induced by these drugs. We will also determine if hERG1 agonists can rescue the function of trafficking-deficient LQTS-associated mutant channels. A molecular-based understanding of the mechanisms of action of known hERG1 activators will enable rational drug design of additional compounds, and facilitate the design of preclinical tests for efficacy and safety. We will utilize biophysical (whole cell voltage clamp, cut-open Vaseline gap voltage clamp), biochemical (Western blot) and modeling techniques to characterize wild-type and mutant hERG1 channels heterologously expressed in Xenopus oocytes and HEK293 cells to characterize the mechanisms of action of four hERG1 channel activators: RPR260263, PD- 118057, NS1643 and ICA-105574. The Aims of the project are to 1) determine binding stoichiometry of hERG1 channel activators, 2) define the mechanism of suppressed channel inactivation by hERG1 channel activators, 3) characterize electromechanical uncoupling induced by hERG1 channel activators, and 4) screen hERG1 activators for ability to rescue trafficking of LQTS-associated mutant channels. It is anticipated that a molecular-based understanding of the mechanisms of action of known hERG1 activators will enable rational drug design of additional compounds, and facilitate the design of preclinical tests for efficacy and safety.